技术资料

Cerebrospinal fluid (CSF) is a vital liquid,providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP),a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here,we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally,the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes. View Publication

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore,the objective was to study the effects of SCFAs on biomarkers of ISC activity,differentiation,barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET),propionate (PROP),or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments,but increased with ACET. As a result of BUT and PROP treatments,there was an increase in differentiation markers for enterocyte,Paneth,goblet,and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3$\beta$,Reg3$\gamma$,and Defb1 were stimulated by BUT and PROP,but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures,although tight junctions were influenced by BUT and PROP,but not ACET in monolayer format. Furthermore,BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall,individual SCFAs are potent stimulators of cellular gene expression,however,PROP and especially BUT show great efficacy for driving cell differentiation and gene expression. View Publication

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells,via single-cell RNA-Seq and protein mass spectrometry,demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro,recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells,with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells,S1P ablation results in an increased production of granulocyte-macrophage progenitors,the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche. View Publication

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones,polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein,respectively,were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence,immunoelectron microscopy and cell surface protein labeling. In addition,measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. View Publication

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation,here,we compared,in multiple cell and in vivo models,mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1m$\Psi$),synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1m$\Psi$ made by the modified process,while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA. View Publication

Periodontitis is an irreversible,bacteria-induced,chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria,neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However,periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation,which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod,Filifactor alocis. With the discovery of these oral bacterial species,there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently,few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus,this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis,cytokine and chemokine signaling pathways,and apoptosis. Moreover,F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient,dampened p38 MAPK activation by secondary stimuli TNF$\alpha$ but not by fMLF. Functionally,the F. alocis-mediated inhibition of p38 activation by TNF$\alpha$ resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNF$\alpha$. Since the modulatory effect was characteristic of viable F. alocis only,we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses. View Publication

The molecular basis of the earliest neuronal changes that lead to Alzheimer's disease (AD) is unclear. Here,we analyze neural cells derived from sporadic AD (SAD),APOE4 gene-edited and control induced pluripotent stem cells (iPSCs). We observe major differences in iPSC-derived neural progenitor (NP) cells and neurons in gene networks related to neuronal differentiation,neurogenesis,and synaptic transmission. The iPSC-derived neural cells from SAD patients exhibit accelerated neural differentiation and reduced progenitor cell renewal. Moreover,a similar phenotype appears in NP cells and cerebral organoids derived from APOE4 iPSCs. Impaired function of the transcriptional repressor REST is strongly implicated in the altered transcriptome and differentiation state. SAD and APOE4 expression result in reduced REST nuclear translocation and chromatin binding,and disruption of the nuclear lamina. Thus,dysregulation of neural gene networks may set in motion the pathologic cascade that leads to AD. View Publication

Upon immunogenic challenge,lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments,and with resolution,they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation,YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease. View Publication

COVID-19 is currently a global pandemic,but human immune responses to the virus remain poorly understood. We analyzed 125 COVID-19 patients,and compared recovered to healthy individuals using high dimensional cytometry. Integrated analysis of {\~{}}200 immune and {\~{}}50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching {\textgreater}30{\%} of circulating B cells. However,another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19." View Publication

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However,it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-$\kappa$B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+),34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV-) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced,single spliced,and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p {\textless} 0.05). As Tat is synthesized by multiple splicing,the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects,which could indicate a higher Tat activity in these cells despite their resting state. View Publication